As recombinant DNA technology has developed in recent years, the controlled production by microorganisms of an enormous variety of useful polypeptides has become possible. Many eukaryotic polypeptides have already been produced by microorganisms and approved by the Food and Drug Administration for pharmeceutical applications, such as for example, human growth hormone, leukocyte interferons, human insulin and human proinsulin. The continued application of techniques already in hand is expected in the future to permit production by microorganisms of a variety of other useful polypeptide products. One such useful polypeptide product is human tumor necrosis factor.
Tumor necrosis factor (TNF) is an antitumor substance found in the sera of animals that have been treated with microbial products in two orderly events. The first event is a priming event that causes the activation and proliferation of macrophages and is associated with expansion of reticuloendothelial elements in the liver and spleen. For this priming event, mycobacteria such as Bacillus Calmette Guerin (BCG), corynebacteria such as Corynebacterium parvum and zymosan (yeast cell walls) are effective. The second event is an elicitation event which is necessary for appearance of TNF in the blood. This requires subsequent treatment of primed animals with lipopolysaccharide (LPS--a major constituent of the cell wall of gram-negative bacteria, also known as endotoxin or bacterial pyrogen). Using these principles, one can obtain sera with similar antitumor and cytotoxic properties from mice, rats and rabbits.
The cellular origin of TNF is macrophages (monocytes), thus TNF is also referred to as a monokine. TNF causes haemorrhagic necrosis and sometimes complete regression of certain tumors transplanted in mice and shows cytotoxic activity against certain tumor cell lines, but not against normal cells. TNF is not species specific, for example, mouse TNF is effective against a wide range of cell lines derived from human cancers.
The gene coding for human TNF has been cloned and expressed in E. coli. Human TNF appears to be encoded by a single copy gene. Four exons code for a precursor product of 233 amino acids and a mature product of 157 amino acids after an unusually long leader sequence has been removed. The molecular weight of human TNF is 45,000 daltons by gel filtration, and, as determined by SDS-PAGE, has a minimum molecular weight of 17,000. TNF has an isoelectric point of 5.6, does not have N-glycosylation sites and has only one possible S-S bridge. Mouse TNF also has been recently cloned and shows approximately 80% homology to human TNF at the (deduced) amino acid level. These recombinant TNF products have biological activities similar to that predicted from work with nonrecombinant TNF.
Recent evidence indicates that TNF belongs to a family of molecules having similar biological activities and varying degrees of sequence homologies. TNF is related in its cytotoxic activity to lymphotoxin. Both of these proteins exhibit synergism with interferon-.gamma. in eliciting cytotoxic activity. TNF shares 30% amino acid homology with lymphotoxin. The single copy genes which encode these proteins share several structural features; each gene is approximately 3 kbp in length and is interrupted by three introns. In addition, these genes are closely linked and both have been mapped to human chromosome 6. In view of these similarities in biological activity, TNF is designated as TNF-.alpha. and lymphotoxin as TNF-.beta.. Although their biological activities are similar, they are derived from different cell types and have distinct induction kinetics. TNF-.alpha. is secreted from monocytes 4-24 hours after induction, while TNF-.beta. is secreted from T-lymphocytes 24-48 hours following induction. Unlike TNF-.alpha., (molecular weight 17,000), TNF-.beta. is a glycosylated protein of monomer molecular weight 25,000. TNF-.alpha. and TNF-.beta. together appear to be the major cytolytic factors produced by peripheral blood leukocytes (PBL). Several lines of evidence suggest that TNF-.alpha. is identical to cachectin, which is one of the principle mediators of the lethal effect of LPS and which is assayed by its ability to suppress the activity of lipoprotein lipase in cultured adipocytes.
In view of the several useful biological activities of TNF with potential commercial value, a method for the production of human TNF-.alpha. with both yields and biological activities superior to that produced in E. coli would be highly desirable.